Conjugated biological molecules and their preparation

ABSTRACT

Novel biologically active compounds of the general formula (I) in which one of X and X′ represents a polymer, and the other represents a hydrogen atom; each Q independently represents a linking group; W represents an electron-withdrawing moiety or a moiety preparable by reduction of an electron-withdrawing moiety; or, if X′ represents a polymer, X-Q-W— together may represent an electron withdrawing group; and in addition, if X represents a polymer, X′ and electron withdrawing group W together with the interjacent atoms may form a ring; each of Z 1  and Z 2  independently represents a group derived from a biological molecule, each of which is linked to A and B via an amine group; or Z 1  and Z 2  together represent a single group derived from a biological molecule which is linked to A and B via two amine groups; A is a C 1-5  alkylene or alkenylene chain; and B is a bond or a C 1-4  alkylene or alkenylene chain; are formed by conjugating a suitable polymer to a suitable biologically active molecule via amine groups in said molecule, preferably via a disulphide bridge.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/077,980,filed Mar. 31, 2011, now U.S. Pat. No. 8,816,051; which is acontinuation of application Ser. No. 12/458,931, filed Jul. 28, 2009,now U.S. Pat. No. 7,939,630; which is a continuation of application Ser.No. 10/564,340, filed Jan. 11, 2006, now U.S. Pat. No. 7,595,292; whichis the U.S. national stage of Application No. PCT/GB2004/02994, filedJul. 12, 2004; the contents of each of which are incorporated byreference herein.

FIELD OF INVENTION

This invention relates to conjugated biological molecules and theirpreparation from novel chemically functionalised derivatives of polymerssuch as polyethylene glycol.

BACKGROUND OF INVENTION

Many therapeutically active molecules do not possess the propertiesrequired to achieve efficacy in clinical medical use. For example,therapeutically active proteins and polypeptides are now beingdiscovered and produced by the biopharmaceutical industry and by geneticengineering. Although there are currently at least 80 protein basedmedicines marketed in the United States with at least 350 more proteinbased medicines undergoing clinical trails (Harris J, Chess R: Effect ofPegylation on pharmaceuticals. Nature Review Drug Discovery, 2003, 2,214-221), most native proteins do not make good medicines because uponadministration to patients there are several inherent drawbacks thatinclude: (1) proteins are digested by many endo and exopeptidasespresent in blood or tissue, (2) many proteins are immunogenic to someextent and (3) proteins can be rapidly excreted by kidneyultrafiltration. Other molecules used as active therapeutic agents inmedicines that are systemically toxic or lack optimal bioavailabilityand pharmacokinetics include low molecular weight molecules where aneffective dose is limited by toxicity. Such molecules are routinely usedto treat inflammation and conditions due to malignancies, infection andautoimmune disease.

Water soluble, synthetic polymers, particularly polyalkylene glycols,are used to conjugate therapeutically active molecules such as proteins.These therapeutic conjugates have been shown to favourably alterpharmacokinetics by prolonging circulation time and decreasing clearancerates, decrease systemic toxicity, and in several cases, to displayincreased clinical efficacy. This process of covalently conjugatingpolyethylene glycol, PEG, to proteins is commonly known as “PEGylation”however many different polymers have been examined as conjugatingpolymers.

Many polymer reagents for conjugation comprise conjugating chemicalfunctionality that is hydrolytically unstable. Examples ofhydrolytically unstable polymeric conjugating reagents are active estersthat include, for example, polyalkylene oxide-N-succinimide carbonates(Zalipsky U.S. Pat. No. 5,122,614). These reagents have relatively shorthalf lives in aqueous media, that includes blood or plasma. This resultsin the need to add large stoichiometric excesses of the conjugatingpolymer reagent. The hydrolytic stability of the reagent is importantbecause the requirement to add stoichiometric excesses for proteinconjugation requires significant effort and cost to purify thepolymer-protein conjugate from the reaction mixture. Furthermore, thesehydrolytically unstable reagents tend to undergo preferentially,reaction with amine chemical functionality in the protein, particularlyto the ε-amine of lysine residues. Since most proteins of interest havemore than one lysine residue, and frequently many lysine residues, thenconjugation tends to be non-specific in that it occurs at many residuesites on the protein. It is possible to purify the conjugating reactionmixture to isolate proteins conjugated to one polymer molecule, howeverit is not possible, to isolate at a reasonable cost, polymer-proteinconjugates that are all conjugated to the same amine group on theprotein. Non-specific conjugation frequently results in impaired proteinfunction. For example antibodies and antibody fragments with randompoly(alkylene oxide) attachment via lysine residues result in modifiedantibodies (or modified antibody fragments) able to bind target antigenwith reduced affinity, avidity or specificity. Additionally, aminespecific polymer conjugating reagents require conjugating reactionconditions that must be selected to ensure that the amines on theprotein are not protonated. These conditions require moderately high pHmedia (8-10), this allows the amine moieties to be reactive enough forreaction with the polymer conjugating reagent. High pH conditions arefrequently deleterious to the protein causing structural changes anddenaturation. These processes result in impairment of protein function.Amine specific polymer conjugation reagents tend to bind to accessibleamine sites on the protein. These reagents can be termed kineticreagents. They are labile and undergo a reaction with the mostassessable amino nucleophilic sites on the protein. Amine specificpolymer conjugating reagents that conjugate by amine acylation result inthe loss of positive charge on the amine group of the amino acid residueon the protein that would normally be present under physiologicalconditions for the unconjugated protein. These features of aminespecific polymer conjugating reagents often leads to partial impairmentof the function of the protein. Other conjugating functional groupsincorporated in polymers for conjugation to protein and that are aminespecific and frequently hydrolytically labile include isocyanate (WO94/04193) and carbonates (WO 90/13540).

Particularly relevant for optimised efficacy and to ensure dose to doseconsistency is to make certain that the number of conjugated polymermolecules per protein is the same and that each polymer molecule isspecifically covalently conjugated to the same amino acid residue ineach protein molecule. Non-specific conjugation at sites along a proteinmolecule results in a distribution of conjugation products andfrequently, unconjugated protein, to give a complex mixture that isdifficult, tedious, and expensive to purify.

Thiol specific polymer conjugating reagents for proteins have beendeveloped to address the limitations for the propensity of theconjugating reagent to undergo hydrolysis that is competitive withconjugation to the protein, non-specific polymer conjugation atdifferent amino acid residues in the protein, and the need for high pHconjugating reaction conditions. Thiol specific polymer conjugatingreagents can be utilised at pH values close to neutrality where theamine functional moieties on the amino acid residues of the protein areprotonated and thus cannot effectively compete in the conjugationreaction with the polymer conjugating reagent. Thiol specific polymerconjugating reagents that are relatively more hydrolytically stable thanare the aforementioned amine specific reagents can be utilised at alower stoichiometric excess thus reducing the cost during purificationof the polymer-protein conjugate. Conjugating functional moieties thatare broadly selective for thiol groups include iodoace-tamide,maleiimide (WO 92/16221), vinylsulfone (WO 95/13312 and WO 95/34326),vinyl pyridines (WO 88/05433), and acrylate and methacrylate esters (WO99/01469). These thiol selective conjugating moieties yield a singlethioether conjugating bond between the polymer.

Most proteins do not have free sulfhydrals because these sulfhydralsundergo rearrangement and scrambling reactions with the disulfidebridges within the protein resulting in impaired protein function. Forproteins that do have free sulfhydrals, these sulfhydrals are frequentlycritical for protein function. Typically in a protein, the number ofsulfhydral moieties is less than the number of amine moieties (e.g.lysine or histadine). Since conjugation to a protein can be made to bespecific at thiol groups and since proteins do not typically have freethiol groups, there are examples of site-specific modification ofprotein by mutagenesis to introduce thiol sites for PEG attachment.However such modifications increase costs significantly. The introducedfree sulhydral can have the similar limitations as mentioned heretoforein the engineered protein for protein scrambling and proteindimerisation. Also the process of mutagensis and production of themodified protein from bacterial sources frequently causes the freesulfhydral to be bound in a disulfide bond with glutathione, forexample. Interleukin-2, for example, has been modified by mutagenesis toreplace a threonine residue by a cysteine to allow site specificattachment of PEG. [Goodson R J, Katre N V; Bio/Technology (1990) 8,343-346].

It is known in the art that conjugating parameters have to be optimallymatched with the therapeutically active molecule of interest in terms ofpolymer morphology, molecular weight characteristics, chemicalfunctionality. Although the polymer protein conjugate, can display manyfavourable and necessary properties needed for safe, effective medicaluse, the effect of polymer conjugation on the activity and stability ofthe protein is of vital importance for performance. Conjugationvariables related to the location and amount of conjugation and polymercharacteristics must be optimally correlated with biological andphysicochemical properties.

SUMMARY OF INVENTION

We have now found a series of novel reagents which can be used interalia to conjugate with both sulphur atoms derived from two cysteineresidues in a protein to give novel thioether conjugates. The inventionin the first instance is intended for the conjugation of the two sulphuratoms that form natural disulphide bridges in native proteins.Disulphide bonds are found in medically relevant proteins, specifically,secretory proteins, lysosomal proteins, and the exoplasmic domains ofmembrane proteins. The technology provides clear advantages over knowntechniques for conjugating polymers to proteins.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the standard curve obtained for different forms ofpolymer-conjugated interferon obtained in Example 7.

FIG. 2 shows a graph of absorbance vs. IFN [pg/ml] obtained for NIBSC(UK) interferon-α 2b (open symbols) and native interferon-α 2b (closedsymbols).

FIG. 3 shows a graph of biological activity vs. IFN [pg/ml] obtained forchemically treated native interferon-α 2b that was not pegylated (opensymbols) and native interferon-α 2b (closed symbols).

FIG. 4 shows a graph of biological activity vs. IFN [pg/ml] obtained forpegylated interferon-α 2b (open symbols) and native interferon-α 2b usedfor conjugation (closed symbols).

FIG. 5A shows real-time quantitative RT-PCR analysis of theinterferon-inducible 2′5′-oligoadenylate synthetase (2′5′-OAS) gene.

FIG. 5B shows real-time quantitative RT-PCR analysis of theinterferon-inducible protein kinase R (PRKR) gene.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention provides a compound of the general formula

-   -   in which one of X and X′ represents a polymer, and the other        represents a hydrogen atom;    -   each Q independently represents a linking group;    -   W represents an electron-withdrawing moiety or a moiety        preparable by reduction of an electron-withdrawing moiety; or,        if X′ represents a polymer, X-Q-W— together may represent an        electron withdrawing group; and in addition, if X represents a        polymer, X′ and electron withdrawing group W together with the        interjacent atoms may form a ring;    -   each of Z¹ and Z² independently represents a group derived from        a biological molecule, each of which is linked to A and B via a        nucleophilic moiety; or Z¹ and Z² together represent a single        group derived from a biological molecule which is linked to A        and B via two nucleophilic moieties;    -   A is a C₁₋₅ alkylene or alkenylene chain; and    -   B is a bond or a C₁₋₄ alkylene or alkenylene chain.

A polymer X or X′ may for example be a polyalkylene glycol, apolyvinylpyrrolidone, a polyacrylate, for example polyacryloylmorpholine, a polyoxazoline, a polyvinylalcohol, a polyacrylamide orpolymethacrylamide, for example polycarboxymethacrylamide, or a HPMAcopolymer. Additionally X or X′ may be a polymer that is susceptible toenzymatic or hydrolytic degradation. Such polymers, for example, includepolyesters, polyacetals, poly(ortho esters), polycarbonates, poly(iminocarbonates), and polyamides, such as poly(amino acids). A polymer X orX′ may be a homopolymer, random copolymer or a structurally definedcopolymer such as a block copolymer. For example X or X′ may be a blockcopolymer derived from two or more alkylene oxides, or frompoly(alkylene oxide) and either a polyester, polyacetal, poly(orthoester), or a poly(amino acid). Polyfunctional polymers that may be usedinclude copolymers of divinylether-maleic anhydride and styrene-maleicanhydride. Naturally occurring polymers may also be used, for examplepolysaccharides such as chitin, dextran, dextrin, chitosan, starch,cellulose, glycogen, poly(sialylic adic) and derivatives thereof.Polymers such as polyglutamic acid may also be used, as may hybridpolymers derived from natural monomers such as saccharides or aminoacids and synthetic monomers such as ethylene oxide or methacrylic acid.

If the polymer is a polyalkylene glycol, this is preferably onecontaining C₂ and/or C₃ units, and is especially a polyethylene glycol.A polymer, particularly a polyalkylene glycol, may contain a singlelinear chain, or it may have branched morphology composed of many chainseither small or large. The so-called Pluronics are an important class ofPEG block copolymers. These are derived from ethylene oxide andpropylene oxide blocks. Substituted polyalkylene glycols, for examplemethoxypolyethylene glycol, may be used. In a preferred embodiment ofthe invention, a single-chain polyethylene glycol is initiated by asuitable group, for example an alkoxy, e.g. methoxy, aryloxy, carboxy orhydroxyl group, and is connected at the other end of the chain to thelinker group Q.

The polymer X or X′ may optionally be derivatised or functionalised inany desired way. For example, polymers with two or more chemicalmoieties for conjugation that are the subject of this invention may beused to create conjugates of two or more linked bioactive molecules.Reactive groups may be linked at the polymer terminus or end group, oralong the polymer chain through pendent linkers; in such case, thepolymer is for example a polyacrylamide, polymethacrylamide,polyacrylate, polymethacrylate, or a maleic anhydride copolymer.Multimeric conjugates that contain more than one biological molecule,typically a biologically active polypeptide or drug can result insynergistic and additive benefits. If desired, the polymer may becoupled to a solid support using conventional methods.

The optimum molecular weight of the polymer will of course depend uponthe intended application. Preferably, the number average molecularweight is in the range of from 500 g/mole to around 75,000 g/mole. Whenthe compound of the general formula I is intended to leave thecirculation and penetrate tissue, for example for use in the treatmentof inflammation caused by malignancy, infection or autoimmune disease,or by trauma, it may be advantageous to use a lower molecular weightpolymer in the range 2000-30,000 g/mole. For applications where thecompound of the general formula I is intended to remain in circulationit may be advantageous to use a higher molecular weight polymer, forexample in the range of 20,000-75,000 g/mole.

The polymer to be used should be selected so the conjugate is soluble inthe solvent medium for its intended use. For biological applications,particularly for diagnostic applications and therapeutic applicationsfor clinical therapeutical administration to a mammal, the conjugatewill be soluble in aqueous media. Many proteins such as enzymes haveutility in industry, for example to catalyze chemical reactions. Forconjugates intended for use in such applications, it may be necessarythat the conjugate be soluble in either or both aqueous and organicmedia. The polymer should not impair the intended function of thebiological molecule(s).

A linking group Q may for example be a direct bond, an alkylene group(preferably a C₁₋₁₀ alkylene group), or an optionally-substituted arylor heteroaryl group, any of which may be terminated or interrupted byone or more oxygen atoms, sulphur atoms, —NR groups (in which R has themeaning given below), keto groups, —O—CO— groups and/or —CO—O— groups.Suitable aryl groups include phenyl and naphthyl groups, while suitableheteroaryl groups include pyridine, pyrrole, furan, pyran, imidazole,pyrazole, oxazole, pyridazine, primidine and purine. The linkage to thepolymer may be by way of a hydrolytically labile bond, or by anon-labile bond.

Substituents which may be present on an optionally substituted aryl orheteroaryl group include for example one or more of the same ordifferent substituents selected from —CN, —NO₂, —CO₂R, —COH, —CH₂OH,—COR, —OR, —OCOR, —OCO₂R, —SR, —SOR, —SO₂R, —NHCOR, —NRCOR, —NHCO₂R,—NR′CO₂R, —NO, —NHOH, —NR′OH, —C═N—NHCOR, —C═N—NR′COR, —N⁺R₃, —N⁺H₃,—N⁺HR₂, —N⁺H₂R, halogen, for example fluorine or chlorine, —C≡CR, —C═CR₂and —C═CHR, in which each R or R′ independently represents a hydrogenatom or an alkyl (preferably C₁₋₆ alkyl) or an aryl (preferably phenyl)group. The presence of electron withdrawing substituents is especiallypreferred.

W may for example represent a keto or aldehyde group CO, an ester group—O—CO— or a sulphone group —SO₂—, or a group obtained by reduction ofsuch a group, e.g. a CH.OH group, an ether group CH.OR, an ester groupCH.O.C(O)R, an amine group CH.NH₂, CH.NHR or CH.NR₂, or an amideCH.NHC(O)R or CH.N(C(O)R)₂. If X-Q-W— together represent an electronwithdrawing group, this group may for example be a cyano group.

In the following portion of this specification, Z¹ and Z² will bereferred to collectively as Z. It is a preferred embodiment of theinvention that Z¹ and Z² together should represent a single biologicalmolecule:

Z may be derived from any desired biological molecule, for example aprotein. The protein may for example be a polypeptide, antibody,antibody fragment, enzyme, cytokine, chemokine or receptor. Constrainedor cyclic polypeptides, which are usually cyclised through a disulphidebridge, and epitopes, may also be used. Specific examples of suitablebiological molecules are listed below.

Preferably a nucleophilic moiety linking A or B to the group(s) Z isderived from a thiol group or amine group. Two thiol groups may begenerated by partial reduction of a natural or engineered disulphide(cysteine) bridge. Amine groups may for example be lysine residues.Where Z¹ and Z² together form a single biological molecule which islinked to the groups A and B via two thiol groups, the compound of theformula I has the formula

The invention also provides a process for the preparation of a compoundof the general formula I, which comprises reacting either (i) a compoundof the general formula

in which one of X and X′ represents a polymer and the other represents ahydrogen atom;

-   -   Q represents a linking group;    -   W′ represents an electron-withdrawing group, for example a keto        group, an ester group —O—CO— or a sulphone group —SO₂—; or, if        X′ represents a polymer, X-Q-W′ together may represent an        electron withdrawing group;    -   A represents a C₁₋₅ alkylene or alkenylene chain;    -   B represents a bond or a C₁₋₄ alkylene or alkenylene chain; and    -   each L independently represents a leaving group;        or (ii) a compound of the general formula

in which X, X′, Q, W′, A and L have the meanings given for the generalformula II, and in addition if X represents a polymer, X′ andelectron-withdrawing group W′ together with the interjacent atoms mayform a ring, and m represents an integer 1 to 4; with a compound of thegeneral formulaZ(Nu)₂  (IV)in which Z has the meaning given above and each Nu independentlyrepresents a nucleophilic group, for example a thiol or an amine group.If Z¹ and Z² are separate molecules, the reaction takes place in twosuccessive steps with successive molecules Z¹Nu and Z²Nu.

The or each leaving group L may for example represent —SR, —SO₂R,—OSO₂R, —N⁺R₃, —N⁺HR₂, —N⁺H₂R, halogen, or —OØ, in which R has themeaning given above, and Ø represents a substituted aryl, especiallyphenyl, group, containing at least one electron withdrawing substituent,for example —CN, —NO₂, —CO₂R, —COH, —CH₂OH, —COR, —OR, —OCOR, —OCO₂R,—SR, —SOR, —SO₂R, —NHCOR, —NRCOR, —NHCO₂R, —NR′CO₂R, —NO, —NHOH, —NR′OH,—C═N—NHCOR, —C═N—NR′COR, —N⁺R₃, —N⁺HR₂, —N⁺H₂R, halogen, especiallychlorine or, especially, fluorine, —C≡CR, —C═CR₂ and —C═CHR, in which Rand R′ have the meanings given above.

Typical structures in which W′ and X′ together form a ring include

in which n is an integer from 1 to 4, and

A compound of the general formula (IV) in which each Nu is a thiol groupmay be prepared by partial reduction of a protein contain a cysteinelink, i.e.

Suitably, the process according to the invention is carried out bypartially reducing a disulfide bond derived from two cysteine aminoacids in the protein in situ following which the reduced product reactswith the compound of formula (II) or (III). Disulfides can be reduced,for example, with dithiothreitiol, mercaptoethanol, ortris-carboxyethylphosphine using conventional methods. The process maybe carried out in a solvent or solvent mixture in which all reactantsare soluble. The biological molecule containing nucleophilic groups(e.g. protein) may be allowed to react directly with the compound of thegeneral formula II or III in an aqueous reaction medium. This reactionmedium may also be buffered, depending on the pH requirements of thenucleophile. The optimum pH for the reaction is generally between about5.5 and about 8, for example about 7.4, preferably about 6.0-6.5.Reaction temperatures between 3-37° C. are generally suitable: proteinsand other biological molecules may decompose or denature impairingfunction if the conjugation reaction is conducted at a temperature wherethese processes may occur. Reactions conducted in organic media (forexample THF, ethyl acetate, acetone) are typically conducted attemperatures up to ambient, for example temperatures below 0° C.

A protein can contain one or a multiplicity of disulfide bridges.Reduction to give free sulfhyral moieties can be conducted to reduce oneor a multiplicity of disulfide bridges in a protein. Depending on theextent of disulfide reduction and the stoichiometry of the polymericconjugation reagent that is used, it is possible to conjugate one or amultiplicity of polymer molecules to the protein. Immobilised reducingagents may be used if it is desired to reduce less than the total numberof disulfides, as can partial reduction using different reactionconditions or the addition of denaturants.

Alternatively the source of the thiol groups can be from cysteines orthiols not originally derived from a disulfide bridge. If the source ofthe thiol groups is a disulfide bridge, this may be intrachain orinterchain.

The biological molecule can be effectively conjugated with the reagentsof the present invention using a stoichiometric equivalent or a slightexcess of reagent, unlike many prior art reagents. However, since thereagents of the present invention do not undergo competitive reactionswith aqueous media used to solvate proteins, it is possible to conductthe conjugation reaction with an excess stoichiometry of reagent. Theexcess reagent can be easily removed by ion exchange chromatographyduring routine purification of proteins.

The compounds of formulae (II) and (III) are novel, and accordingly theinvention further provides these compounds per se. These novel reagentsprovide a breakthrough in conjugate technology, the chemical functionalmoiety on the polymer comprising a cross-functionalised, latentlycross-conjugated, bis-alkylating moiety that is selective for twonucleophiles, particularly two thiols derived from a natural disulfidebond in proteins.

The immediate product of the process according to the invention is acompound of the general formula I in which W is an electron-withdrawinggroup. Such compounds have utility in themselves; because the process ofthe invention is reversible under suitable conditions, additionallycompounds of formula (I) in which W is an electron-withdrawing moietyhave utility in applications where release of the free protein isrequired, for example in direct clinical applications. Anelectron-withdrawing moiety W may, however, be reduced to give a moietywhich prevents release of the protein, and such compounds will also haveutility in many clinical, industrial and diagnostic applications.

Thus, for example, a moiety W containing a keto group may be reduced toa moiety W containing a CH(OH) group; an ether group CH.OR may beobtained by the reaction of a hydroxy group with an etherifying agent;an ester group CH.O.C(O)R may be obtained by the reaction of a hydroxygroup with an acylating agent; an amine group CH.NH₂, CH.NHR or CH.NR₂may be prepared from a ketone or aldehyde by reductive amination); or anamide CH.NHC(O)R or CH.N(C(O)R)₂ may be formed by acylation of anamine). A group X-Q-W— which is a cyano group may be reduced to an aminegroup.

A compound of the general formula (II) in which X represents a polymermay be prepared by reacting a compound of the general formula

-   -   in which Q′, W, A, B and L have the meanings given above, with a        polymer of the general formula        X—V  (VII)        in which X represents a polymer; Q′ and V being groups selected        such that the compounds of (VI) and (VII) will react together to        give the desired compound of the formula (II). Alternatively, a        compound of the formula

may be reacted with a polymer of the general formulaX-Q′  (IX)

A compound of the general formula (III) may be prepared by base mediatedelimination of one leaving group L from a compound of the generalformula (II).

Compounds of the general formula I may include an imaging agent, forexample a radio nucleotide, to enable tracking of the compound in vivo.Suitably the radio nucleotide or imaging agent I may be bound throughthe group W, to give, for example, compounds of the type

which may for example be prepared from reagents of the type

for example

The compounds of the general formula I have a number of applications.They may for example be used for direct clinical application to apatient, and accordingly, the present invention further provides apharmaceutical composition comprising a compound of the general formulaI together with a pharmaceutically acceptable carrier. The inventionfurther provides a compound of the general formula I for use as amedicament, and a method of treating a patient which comprisesadministering a pharmaceutically-effective amount of a compound of theformula I or a pharmaceutical composition according to the invention tothe patient. Any desired pharmaceutical effect, for example traumatreatment, enzyme replacement, toxin removal, anti-inflammatory,anti-infective, immunomodulatory, vaccination or anti-cancer, may beobtained by suitable choice of biological molecule.

The compounds of the general formula I may also be used in non-clinicalapplications. For example, many physiologically active compounds such asenzymes are able to catalyse reactions in organic solvents, andcompounds of the general formula I may be used in such applications.Further, compounds of the general formula I may be used as diagnostictools.

The following gives some specific biological molecules which may haveutility in the present invention, depending upon the desiredapplication. Enzymes include carbohydrate-specific enzymes, proteolyticenzymes and the like. Enzymes of interest, for both industrial (organicbased reactions) and biological applications in general and therapeuticapplications in particular include the oxidoreductases, transferases,hydrolases, lyases, isomerases and ligases disclosed by U.S. Pat. No.4,179,337. Specific enzymes of interest include asparaginase, arginase,adenosine deaminase, superoxide dismutase, catalase, chymotrypsin,lipase, uricase, bilirubin osidase, glucose oxidase, glucuronidase,galactosidase, glucocerbrosidase, glucuronidase, glutaminase

The biologically active molecules used in compounds of the generalformula I of the present invention include for example factor 8,insulin, ACTH, glucagen, somatostatin, somatotropins, thymosin,parathyroid hormone, pigmentary hormones, somatomedins, erythropoietin,luteinizing hormone, hypothalamic releasing factors, antidiuretichormones, prolactin, interleukins, interferons, colony stimulatingfactors, hemoglobin, cytokines, antibodies, chorionicgonadotropin,follicle-stimulating hormone, thyroid stimulating hormone and tissueplasminogen activator.

Certain of the above proteins such as the interleukins, interferons andcolony stimulating factors also exist in non-glycosilated form, usuallythe result of preparation by recombinant protein techniques. Thenon-glycosilated versions may be used in the present invention.

For example, for Interferons, the invention permits the preparation ofconjugates in which the biological activity is retained compared withnon-conjugated Interferons. This most surprising result is not possibleusing known conjugation techniques.

Other proteins of interest are allergen proteins disclosed by Dreborg etall Crit. Rev. Therap. Drug Carrier Syst. (1990) 6 315 365 as havingreduced allergenicity when conjugated with a polymer such aspoly(alkylene oxide) and consequently are suitable for use as toleranceinducers. Among the allergens disclosed are Ragweed antigen E, honeybeevenom, mite allergen and the like.

Glycopolypeptides such as immunoglobulins, ovalbumin, lipase,glucocerebrosidase, lectins, tissue plasminogen activator andglycosilated inerleukins, interferons and colony stimulating factors areof interest, as are immunoglobulins such as IgG, IgE, IgM, IgA, IgD andfragments thereof.

Of particular interest are antibodies and antibody fragments which areused in clinical medicine for diagnostic and therapeutic purposes. Theantibody may used alone or may be covalently conjugated (“loaded”) withanother atom or molecule such as a radioisotope or acytotoxic/antiinfective drug. Epitopes may be used for vaccination toproduce an immunogenic polymer—protein conjugate.

A key feature of the process of the invention is that an α-methyleneleaving group and a double bond are cross-conjugated with an electronwithdrawing function that serves as a Michael activating moiety. If theleaving group is prone to elimination in the cross-functional reagentrather than to direct displacement and the electron-withdrawing group isa suitable activating moiety for the Michael reaction then sequentialintramolecular bis-alkylation can occur by consecutive Michael and retroMichael reactions. The leaving moiety serves to mask a latent conjugateddouble bond that is not exposed until after the first alkylation hasoccurred and bis-alkylation results from sequential and interactiveMichael and retro-Michael reactions as described in J. Am. Chem. Soc.1979, 101, 3098-3110 and J. Am. Chem. Soc. 1988, 110, 5211-5212.). Theelectron withdrawing group and the leaving group are optimally selectedso bis-alkylation can occur by sequential Michael and retro-Michaelreactions.

It is also possible to prepare cross-functional alkylating agents withadditional multiple bonds conjugated to the double bond or between theleaving group and the electron withdrawing group as described in J. Am.Chem. Soc. 1988, 110, 5211-5212.

Since the cross-functionalised bis-alkylating reagents of the typementioned above undergo alkylation that is controlled by Michael-retroMichael equilibria and since it is possible to partially reduce a numberfrom one to a greater than one disulfide bonds in a protein in acontrolled fashion significantly retaining tertiary structure, it isthen possible to have bis-alkylation occur across the two sulfhydralsfrom cysteines of a given disulfide bond. Such a sequence of reactionsresults in the reannealling of the disulfide bridge with thebis-alkylating reagent.

The thiol ether bonds formed upon conjugation to give a compound of theformula I are, in general, hydrolytically stable in aqueous solution.The reagents themselves are also hydrolytically stable. In this context,a compound is regarded as being hydrolytically stable if it does notundergo substantial degradation at physiological pH and temperature upto 45° C. Degradation of less than 50% under these conditions over aneight hour period is considered insubstantial.

It will be appreciated that this invention allows the production ofpolymer reagents that possess cross-functionalised bis-alkylatingfunctionality at either the termini or on pendent chains along the mainchain of a polymer.

Some examples of novel conjugates according to the invention include thefollowing:

Some examples of novel reagents according to the invention include thefollowing:

The following Examples illustrate the invention. FIGS. 1 to 5 show theresults obtained from Example 7.

EXAMPLE 1 Synthesis of Polymer Conjugating Reagentp-Nitro-3-piperidinopropriophenone Hydrochloride: C₁₄H₁₉ClN₂O₃

To a single-neck 250 ml round-bottom flask was added p-nitroacetophenone(16.5 g), paraformaldehyde (4.5 g), piperidine hydrochloride (12.1 g),absolute ethanol (100 mL) and a magnetic stir bar. To the stirredheterogeneous mixture was added hydrochloric acid (37 wt % in water, 1mL) and the solution was heated to reflux under nitrogen. After a 1-2 hperiod more paraformaldehyde (3.0 g) was added. The solution was allowedto reflux for approximately 18 h during which time furtherparaformaldehyde was added (3.0 g). After allowing the reaction solutionto cool a crystalline solid settled that would not dissolve upon furtherrefluxing. The solid was isolated by filtration and recrystallised usingvery hot methanol to afford large yellow crystals (10.9 g). ¹H NMR(DMSO-d₆) δ 1.34-1.50 (m, 1H), 1.64-1.79 (m, 2H), 1.79-1.94 (m, 4H),2.89-3.05 (m, 2H), 3.41 (q, 2H), 3.51-3.54 (m, 2H), 3.82 (t, 2H), 8.26(s, 1H), 8.29 (s, 1H), 8.41 (s, 1H), 8.44 (s, 1H).

3-(2-Hydroxyethylthiol)-p-nitropropiophenone

A stirred solution of p-nitro-3-piperidinopropiophenone hydrochloride(30 g, 0.1 mol) and mercaptoethanol (9.5 g 0.12 mol) in 95% ethanol (200ml) was slowly heated until homogeneous. Piperidine (1.0 ml) was addedand the reaction mixture heated to reflux for 2 h. After cooling themajority of the solvent was rotoevaporated and ethyl acetate (200 ml)added and the solid filtered. The ethyl acetate solution was extractedsequentially with 10% aqueous HCl, 5% NaHCO₃ and brine, and then driedover Na₂SO₄, and then the ethyl acetate was evaporated to an oil thatcrystallised to give 23.2 g of the desired product that wasrecrystallised in ethyl acetate-ethyl ether.

2,2-Bis(p-tolylthiomethyl)-p-nitroacetophenone: C₂₄H₂₃NO₃S₂

To a 100 ml single-neck round-bottom flask was addedp-nitro-3-piperidinopropiophenone hydrochloride (10.0 g),4-methylbenzenethiol (8.2 g), formaldehyde (37% w/w aq. solution, 10 ml,excess), methanol (40 ml) and a magnetic stir bar. The stirredheterogeneous mixture was heated until a yellow homogeneous solutionformed (a couple of minutes at 50-60° C.). Five drops of piperidine werethen added and the reaction solution heated to reflux. Within 15 min thereaction became heterogeneous due to the presence of some white/yellowsolid and after 2 h this solid became a strong orange in colour. Therefluxing was stopped after this time and the reaction was allowed tocool overnight to room temperature. The reaction mixture was then heatedunder reflux again with additional formaldehyde (37% w/w aq. solution,10 ml, excess). After refluxing for approximately 30 min an orange oilwas visible and no solid. The oil would settle to the bottom of theflask when the stirring was stopped. After a further 7 h of refluxing,the mixture was allowed to cool overnight, which resulted in the settledoil crystallising. The crystalline solid was isolated and purified byrecrystallisation from very hot methanol with several drops of acetoneadded to afford yellow crystals (10.0 g). ¹H NMR (DMSO-d₆) δ 2.31 (s,6H), 3.31-3.33 (m, 4H), 3.97 (quintet, 1H), 7.14 (q, 8H), 7.80 (d, 2H),8.24 (d, 2H).

2,2-Bis(p-tolylsulfonylmethyl)-p-nitroacetophenone: C₂₄H₂₃NO₇S₂

In a 250 ml round-bottom flask a suspension of2,2-bis(p-tolylthiolmethyl)-p-nitroacetophenone (2.5 g) and Oxone (18.4g) was stirred in a 1:1 methanol:water (100 ml) for 16 h. This affordeda white solid suspension, to which was added chloroform (100 ml) and theresulting organic phase was isolated using a separating funnel to leavea white solid suspension within the aqueous phase. Additional water wasadded to the aqueous phase until a homogeneous solution formed, whichwas then washed again with chloroform (100 ml). The organic phases werecombined, washed with brine (50 mL×2), dried with magnesium sulfate andthe solvent removed affording an off-white crude solid product afterdrying in vacuo (2.5 g). The product was recrystallised from acetone togive white crystals. ¹H NMR (CDCl₃) δ 3.43-3.62 (m, 4H), 4.44 (quintet,1H), 7.35 (d, 4H), 7.68 (d, 4H); 7.88 (d, 2H), 8.22 (d, 2H); analysiscalculated for C₂₄H₂₃NO₇S₂ (found): C, 57.47 (57.27); H, 4.62 (4.74); N2.79 (2.58); MS (FAB) m/z 502 ([M+1]⁺).

2-(2-Hydroxyethylsulfonylmethyl)-p-nitro-2(Z), 4-penta-dienophenone

To a flame dried round bottom flask purged with argon and fitted with athermometer and dropping funnel was added3-(2-hydroxyethylthiol)-p-nitropropiophenone (0.5 g, 2.0 mmol) andanhydrous tetrahydrofuran (50.0 ml). The solution was stirred and cooledby a dry ice-acetone bath, then TiCl₄ (0.23 ml, 2.1 mmol) was added bysyringe. The ice bath was removed and the solution allowed to warm toambient temperature, then the solution was cooled to −40° C. anddiisopropylethyl amine (1.1 ml) was added by syringe. The cooling bathwas removed and the reaction mixture allowed to warm to −15 to 0° C.turning a red colour. The reaction mixture was then warmed to 25° C.over a 3-5 minute period and a solution of acrolein (0.14 g, 2.1 mmol)in anhydrous tetrahydrofuran (20 ml) was added by dropping funnel over a30-40 minute period. The exothermic reaction caused the temperature ofthe reaction mixture to increase to 30-40° C. and the solution stirred afurther 20 minutes after addition of the aldehyde before addition ofethyl acetate (75 ml). Thin layer chromatography was used to confirm thedisappearance of the starting3-(2-hydroxyethylthiol)-p-nitropropiophenone and the formation of thedesired 2-(2-hydroxyethylthiomethyl)-p-nitro-2(Z), 4 pentadienophenone(R_(f)˜0.38-0.45). The minor product (E-isomer) could be observed at alower R_(f) in the range of 0.29-0.34. The ethyl acetate reactionmixture was extracted with 10% aqueous HCl and brine. The aqueous layerswere combined and extracted twice with ethyl acetate and all the ethylacetate fractions were combined and washed twice with 10% aqueous HCl,5% aqueous NaHCO₃ and brine, then dried over solid Na₂SO₄. The ethylacetate was rotoevaporated to give the crude2-(2-hydroxyethylthiomethyl)-p-nitro-2(Z), 4 pentadienophenone as an oilthat was immediately oxidised as for the synthesis of2,2-bis[(p-tolylsulfonylmethyl)]-p-nitroacetophenone to give2-(2-hydroxyethylsulfonylmethyl)-p-nitro-2(Z), 4-pentadienophenone as asolid that was purified by column chromatography or recrystallised inethyl acetate or methanol and then covalently bound to amino terminatedpoly(ethylene glycol) as described elsewhere in this specification.

The above sequence of reactions was conducted with many aldehydesincluding acealdehyde, methacrolein, ethacrolein butryaldehyde,crotonaldehyde, 2,4-pentadienyal, sorb-aldehyde, tolualdehyde,cinnamaldehyde, methyl-cinnamaldehyde, chloro-cinnamaldehyde,5-phenyl-2,4 pentadienal and 7-phenyl-2,4,6-heptatrienal. This sequenceof reactions was conducted with many aryl keto derivatives including3-(p-tolylthiomethyl)-m-nitroacetophenone,3-(2-hydroxyethylthio)-m-nitropropriophenone,3-(ethylthiomethyl)-m-nitropropiophenone,3-(dimethylaminoethylthio)-m-nitropropiophenone,3-(2-hydroxyethylthio)-3-phenylpropriophenone,3(2-hydroxyethylthiol)-5-phenyl-4(E)-pentenophenone,3(ethylthiomethyl-o-nitropropiophenone, 3-(ethylthio)-propiophenone,3-(2-hydroxyethylsulfonyl)propiophenone,2-(3-(2-hydroxyethylthio)-1-propenyl)-m-2(E)-4-pentadienophenone. Thissequence of reactions was also conducted with aliphatic keto derivativesincluding 4-(ethylthio)-2-butanone, 4-(-p-tolylthio)-2-butanone,4-(4-nitrophenylthio)-2-butanone, 4-(2-hydroxyethylthio)-2-butanone andmethyl-3-(2-hydroxyethylthiol)-propanoate. The final products of theaforementioned reactions for the these precursors can be covalentlybound to poly(ethylene glycol) as described elsewhere in thisspecification.

2,2-Bis[(p-tolylsulfonyl)methyl]-p-aminoacetophenone hydrochlorideC₂₄H₂₆ClNO₅S₂

To a 100 mL round bottom flask was added2,2-bis[(p-tolylsulfonyl)methyl]-p-nitroacetophenone (2 g), ethanol (25mL), hydrochloric acid (37 wt. % in water, 8 mL) and a magnetic stirbar. To the resulting heterogeneous mixture was then added tin(II)chloride dihydrate and the mixture heated in an oil bath at 45° C. for 2h. Water was then added to the homogeneous yellow solution that hadformed to a point where it appeared that a precipitation may occur ifmore water was added. The homogenous solution was allowed to cool toroom temperature resulting in a yellow compoundcrystallising/precipitating out, which was isolated by filtration undervacuum. The isolated product was then mixed with a heated mixture ofacetone and methanol (approximately 90:10 v/v). An insoluble solid wasisolated by filtration under vacuum and dried to constant mass in avacuum oven (1.4 g). ¹H NMR (DMSO-d₆) δ 2.50 (s, 6H), 3.57-3.73(overlapping m's, 5H), 6.27 (s, 2H), 6.39 (d, 2H), 6.96 (d, 2H), 7.47(d, 4H), 7.55 (d, 4H).

Coupling of 2,2-bis[(p-tolylsulfonyl)methyl]-p-aminoacetophenone toα-methoxy-ω-amino poly(ethylene glycol)

A one neck 100 mL round bottom flask fitted with a dropping funnel andnitrogen line was charged with triphosgene (23 mg),2,2-bis[(p-tolylsulfonyl)methyl]-p-aminoacetophenone hydrochloride (125mg), anhydrous toluene (2.5 mL) and a magnetic stir bar under a nitrogenatmosphere. The dropping funnel was separately charged with anhydroustriethylamine (68 μL) and anhydrous toluene (2.5 mL). An acetone/dry-icebath was placed below the round bottom flask and the contents allowed tocool. The triethylamine solution was then added dropwise to thetriphosgene solution under stirring over 5-10 min. The flask and dry-icebath were allowed to warm to room temperature which took several hoursand once at room temperature the reaction mixture was allowed to furtherstir for about 2 h, still under a nitrogen atmosphere. A solution ofO-(2-aminoethyl)-O′-methylpoly(ethylene glycol) 2,000 (490 mg) andanhydrous triethylamine (68 μL) in anhydrous toluene was then addeddropwise to the reaction mixture at room temperature. The resultingmixture was allowed to stir at room temperature overnight (approximately20 h total). The reaction mixture was then opened to the atmosphere andfiltered under gravity through a 5 mL disposable syringe with a piece ofnon-absorbent cotton wool to act as a filter. The homogeneous eluent wastransferred to a 100 mL separating funnel and then washed twice withdeionised water (30 mL and then 10 mL). The aqueous phases were combinedand then washed with diethyl ether (approximately 25 mL). The aqueousphase was then freeze-dried to give an off-white solid product (160 mg).Product was also found in diethyl ether and toluene phases. ¹H NMR(CDCl₃) δ 2.5 (s), 3.39 (s), 3.41-3.53 (overlapping m's), 3.53-3.76 (m),3.82 (t), 4.17 (quintet), 7.35-7.39 (m, 1.39), 7.46 (d), 7.68 (d).

EXAMPLE 2 Synthesis of Polymer Conjugating Reagentp-Carboxy-3-piperidinopropriophenone hydrochloride

To a 250 mL single-neck round-bottom flask was added p-acetyl benzoicacid (10 g) and piperidine hydrochloride (7.4 g), 100 mL of absoluteethanol and a magnetic stir bar. To the stirred heterogeneous mixturewas added concentrated hydrochloric acid (1 mL) and the solution wasthen heated to reflux under nitrogen. Paraformaldehyde (3.7 g) was addedto the flask and refluxing continued for approximately 1.5 h. Ahomogeneous solution formed to which was added more paraformaldehyde(3.7 g). Heating was continued for approximately 6 h during which timefurther paraformaldehyde was added (3.7 g.). The reaction solution wasallowed to cool to room temperature and left for 1 week. A white solidwas isolated by filtration of the cooled reaction mixture. An attemptwas made to crystallise the solid after dissolving in very hot methanol.An insoluble product (1.96 g) was isolated by filtration and a secondproduct (0.88 g) crystallised out of the filtrate. Both productsappeared identical by infra-red spectroscopy and thin layerchromatography analysis after drying in-vacuo and so were combined to beused in subsequent reactions. ATR-FT-IR 1704, 1691, 1235, 760.

4-[2,2-Bis[(p-tolylthio)methyl]acetyl]benzoic acid: C₂₅H₂₄O₃S₂

To a 50 mL single-neck round-bottom flask was addedp-carboxy-3-piperidinopropriophenone hydrochloride (2.5 g),4-methylbenzenethiol (2.1 g) formaldehyde (37% w/w aq. solution, 2.5mL), ethanol (10 mL), a magnetic stir bar and piperidine (approximatelyten drops). A condenser was then fitted to the flask and the reactionsolution heated to reflux. Methanol (5 mL) was then added. After about 2h, additional formaldehyde (2.5 mL) was added and the heating continuedfor a further 2 h. The reaction flask was then allowed to cool to roomtemperature whereupon the reaction solution was diluted with diethylether (approximately 150 mL). The resulting organic phase was thenwashed with water (acidified using 1N hydrochloric acid to pH 2-3; 50mL×2), water (50 mL) and brine (75 mL) and then dried over magnesiumsulfate. Filtration followed by removal of volatiles on a rotaryevaporator afforded a solid residue. The solid was dissolved in aminimum volume of a mixture of predominately methanol and acetone withheating. The homogeneous solution was then placed in a freezerovernight, affording off-white crystals, which were isolated byfiltration under vacuum, washed with fresh acetone and then dried toconstant mass in a vacuum oven (2.5 g): ¹H NMR (CDCl₃) δ 2.38 (s, 6H),3.16-3.31 (m, 4H), 3.85 (quintet, 1H), 7.15 (d, 4H), 7.18 (d, 4H), 7.64(d, 2H), 8.07 (d, 2H); analysis calculated for C₂₅H₂₄O₃S₂ (found): C,68.78 (68.84); H, 5.54 (5.77).

4-[2,2-Bis[(p-tolylsulfonyl)methyl]acetyl]benzoic acid C₂₅H₂₄O₇S₂

In a 250 mL round-bottom flask a suspension of4-[2,2-bis[(p-tolylthio)methyl]acetyl]benzoic acid (2 g) and Oxone (16.9g) was stirred in 1:1 v/v methanol:water (100 mL) for 16 h. Thisafforded a white solid suspension to which was added chloroform (100 mL)and the resulting organic phase was isolated using a separating funnelto leave a white solid suspension with the aqueous phase. To theheterogenous aqueous phase was added additional water until homogenous(approximately 170 mL) and then the aqueous phase was washed withchloroform (75 mL). The organic phases were combined, washed with water(50 mL×2, acidified with a few drops of 1N hydrochloric acid) and brine(50 mL). The organic phase was dried with magnesium sulfate, filteredand the solvent removed using a rotary evaporator to give an off-whitecrude solid product (2.2 g). Further purification (on 1 g of product)was performed by recrystallisation from very hot ethyl acetate, acetoneand hexane affording 0.6 g of product: ¹H NMR (CDCl₃) δ 2.51 (s, 6H),3.49-3.72 (m, 4H), 4.44 (quintet, 1H), 7.40 (d, 4H), 7.73-7.78 (m, 6H),8.13 (d, 2H); analysis calculated for C₂₅H₂₄O₇S₂ (found): C, 59.98(59.88); H, 4.83 (4.78).

Coupling of 4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzoic acid toα-methoxy-ω-amino PEG (2000 g/mol)

A one-neck 50 mL schlenk flask was charged with4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (100 mg) and amagnetic stir bar. The flask was sealed and a strong vacuum applied forapproximately 15 min. An argon atmosphere was introduced into the flaskand thionyl chloride (1 mL) added by syringe with stirring. Theresulting mixture was heated at 50° C. for 2 h. Volatile liquids werethen removed in vacuum to afford a yellow foam. An argon atmosphere wasagain introduced to the flask and anhydrous dichloromethane (5 mL) wasadded by syringe to afford a homogeneous solution. Volatile liquids werethen removed again in vacuum. The solvent addition/removal process wasrepeated until a white residue remained. Anhydrous dichloromethane (5mL) was again added to the schlenk flask to form a homogeneous solution.Separately in a 25 mL round bottom flask fitted with a septum and with amagnetic stir bar, O-(2-aminoethyl)-O′-methylpoly(ethylene glycol)(2,000 g/mol) (0.2 g), and anhydrous triethylamine (30 μL) weredissolved in anhydrous dichloromethane (5 mL) under an argon atmosphere.The reaction solution with bis[(p-tolylsulfonyl)-methyl]acetyl]benzoicacid was injected into the flask containing the poly(ethylene glycol)solution in a dropwise fashion, immediately resulting in the evolutionof a white gas. The resulting solution was allowed to stir overnight atroom temperature whereupon additional triethylamine (28 μL, was added).After a further 1 h, the reaction solution was added dropwise through aglass pipette into rapidly stirred diethyl ether. To achieveprecipitation it was necessary to add hexane to the diethyl ethersolution and place the flask in an ice bath. The precipitant obtainedwas isolated by centrifugation and dried in a vacuum oven to constantweight affording an off-white solid product (230 mg). Furtherpurification was achieved by precipitation by first dissolving theproduct in dichloromethane and adding to a chilled stirred diethyl ethersolution. ¹H NMR (CDCl₃) δ 2.51 (s), 3.40 (s), 3.60-3.75 (m), 3.84 (t),4.36 (quintet), 7.39 (d), 7.68 (d), 7.71 (d), 7.85 (d).

Coupling of 4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzoic acid toα-methoxy-ω-amino PEG (20,000 g/mol)

A one-neck 50 mL Schlenk flask was charged with4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid (100 mg) and amagnetic stir bar. The flask was sealed and a strong vacuum applied forapproximately 15 min. An argon atmosphere was introduced into the flaskand thionyl chloride (1 mL) added by syringe with stirring. Theresulting mixture was heated at 50° C. for 2 h. After allowing the flaskto cool to room temperature, volatile liquids were removed in vacuum toafford a yellow foam. An argon atmosphere was again introduced to theflask and anhydrous dichloromethane (5 mL) was added by syringe toafford a homogeneous solution. Volatile liquids were then removed againin vacuum. The solvent addition-removal process was repeated until awhite residue remained. Finally, anhydrous dichloromethane (5 mL) wasadded to the flask under argon to form a homogeneous solution.

In a separate 50 mL Schlenk flask,O-(2-aminoethyl)-O′-methylpoly(ethylene glycol) (20,000 g/mol) (0.5 g)was dissolved in 4 mL of anhydrous toluene in an argon atmosphere andthen the solution was evaporated to dryness in vacuum. The white residueafforded was kept in vacuum for 3 h and then dissolved in anhydrousdichloromethane (5 mL) in an argon atmosphere.

To the poly(ethylene glycol) solution, which was stirred and cooled inan ice-water bath, was slowly added the reaction solution ofbis[(p-tolylsulfonyl)-methyl]acetyl]benzoic acid drop-wise. Aftercomplete addition 40 μL of dried triethylamine was slowly added to theresulting solution drop-wise. When the triethylamine had been added theresulting solution was allowed to stir overnight and warm to roomtemperature.

The reaction solution was then filtered through a 0.45 μm filter andvolatile liquids removed from the eluent to afford a slightlyyellow/orange residue that was redissolved in warm acetone (10 mL). Theflask containing the resulting homogeneous solution was placed in anice-water bath and under stirring a precipitation occurred. The whiteprecipitate was isolated on a no. 3 sintered glass funnel and washedwith chilled fresh acetone (approximately 30 mL). The isolated solid wasallowed to dry in vacuum to afford 0.4 g of white solid. ¹H NMR (CDCl₃)δ 2.42 (s), 3.31 (s), 3.40-3.75 (m), 4.27 (m), 7.30 (d), 7.59 (d), 7.63(d), 7.75 (d).

The α-methoxy-ω-4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzamidepoly(ethylene glycol) conjugating reagent (20,000 g/mol) (0.4 g) wasdissolved in 10 mL of an argon purged mixture of acetonitrile:water(24:1 v/v, 2 mg hydroquinone) and the solution filtered through a 0.45μm PP filter. The eluent was placed under argon in a round-bottom flaskand triethylamine (50 μL) was added under stirring. The flask was thenplaced in a 30° C. oil bath for 20 h with stirring after which time theflask was cooled to room temperature and the volatile liquids removed invacuum. The residue obtained was dissolved in warm acetone (10 mL) andonce homogeneous the solution was placed in an ice-water bath whereuponprecipitation occurred while the solution stirred. The white precipitatewas isolated on a no. 3 sintered glass funnel and washed with chilledfresh acetone (approximately 30 mL). The isolated solid was allowed todry under vacuum to afford 0.3 g of white solid. ¹H NMR (CDCl₃) δ 2.35(s), 3.31 (s), 3.39-3.78 (m), 4.28 (s), 5.84 (s), 6.22 (s), 7.26 (d),7.65 (d), 7.72 (d), 7.81 (d)

EXAMPLE 3 Reaction of 4-methylbenzenethiol andα-methoxy-ω-4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzamidepoly(ethylene glycol)

The polymer conjugating reagent,α-methoxy-ω-4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzamidepoly(ethylene glycol) (2000 g/mol) (30 mg, 12.1 μmol, 1 eq.) and4-methylbenzenethiol (3 mg, 24.2 mmol, 2 eq.) were dissolved indeuterated chloroform (approximately 0.75 mL). To the homogeneoussolution was then added triethylamine (1.7 μL, 12.1 μmol, 1 eq.). Thereaction mixture was stirred and a H-NMR spectrum was obtained. Theresulting spectrum confirmed that addition of the 4-methylbenzenethiolto the polymer conjugating reagent had occurred. A similar reaction wasconducted with propane triol.

EXAMPLE 4 Polymer Conjugation to Ribonuclease A

To Ribonuclease A (30 mg) in a 15 mL centrifuge tube was added 3 mL ofan 8M aqueous urea solution, followed by 2-mercaptoethanol (60 μL). ThepH of the resulting solution was adjusted to pH 8.5 using a 10% aqueoussolution of methylamine. The reaction solution was then bubbled withnitrogen for approximately 30 min. Still purging with nitrogen the tubewas heated at 37° C. for 5 h. The reaction mixture was then cooled in anice-salt water bath and 10 mL of an argon purged chilled solution of 1NHCl:absolute ethanol (1:39 v/v) was added to the reaction solution. Aprecipitation occurred and the precipitate was isolated bycentrifugation and then washed three times with further 10 mL portionsof the HCl:absolute ethanol mixture and twice with nitrogen purgedchilled diethyl ether (2×10 mL). After each washing the precipitate wasisolated by centrifugation. The washed precipitate was then dissolved innitrogen purged deionised water and freeze-dried to afford a dry solid.Partial reduction of Ribonuclease A was confirmed and quantitated usingEllman's Test, which gave 5.9 free thiols per protein molecule.

In an eppendorf, the partially reduced Ribonuclease A (10.9 mg) wasdissolved in argon purged pH 8 ammonia solution (500 μL). In a separateeppendorf, the polymer conjugating reagent,α-methoxy-ω-4-[2,2-bis[(p-tolylsulfonyl)-methyl]acetyl]benzamide derivedfrom poly(ethylene)glycol (2000 g/mol) (5 mg) was also dissolved inammonia solution (250 μL) and the resulting solution added to theRibonuclease A solution. The PEG eppendorf was washed with 250 μL offresh ammonia solution and this was also added to the main reactioneppendorf. The reaction eppendorf was then closed under argon and heatedat 37° C. for approximately 24 h and then allowed to cool to roomtemperature. The cooled reaction solution was then analysed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TheSDS-PAGE experiment was consistent with reaction of pegylated4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]-benzoic acid withRibonuclease A.

EXAMPLE 5 Polymer Conjugation to Mouse IgG Antibody Fab Fragment

To 240 μL of a 0.4 mg/mL solution of mouse IgG antibody Fab fragment(Abcam Cat. No. AB6668) in an argon purged 0.02 M sodium phosphate pH 6buffer containing 0.15 M sodium chloride and 0.005 M EDTA was added 4 μLof an argon purged aqueous solution of 1 mM selenocystaminedihydrochloride, followed by 12 μL of an argon purged aqueous solutionof tris(2-carboxyethyl) phosphine hydrochloride (TCEP) at roomtemperature. The resulting solution was immediately vortexed for severalseconds and then allowed to stand at room temperature for 6 min. Two 5μL samples of the solution containing reduced Fab fragment were removedfor future analysis and the remaining solution was then allowed to standin an ice-water bath for 4 min. An ice-water bath chilled solution ofα-methoxy-ω-4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzamide derivedfrom poly(ethylene glycol) (20,000 g/mol) (1.6 μL, 50 mg/ml, argonpurged 0.02 M sodium phosphate pH 6 buffer containing 0.15 M sodiumchloride, 0.005 M EDTA and 0.23 mM hydroquinone) was immediately addedto the Fab solution; the solution vortexed for several seconds and thenplaced back into the ice-water bath. A further 1.6 μL of the conjugationreagent solution was added in an analogous manner every two minutesuntil 6.4 μL had been added in total. After complete addition thereaction solution was placed in a refrigerator (<5° C.) for 2 h. Asample of the reaction solution was then removed for analysis by SDSPAGE.

The reaction mixture was analysed by SEC-HPLC (100 μl injection,superpose 12-24 ml column, Amersham biosciences; eluent: 20 mM NaPhosphate, 0.15 M NaCl pH 7.0; isocratic elution for 100 min at a flowof 0.25 ml/min; UV detection, 210 nm) displaying the Fab-poly(ethyleneglycol) conjugate that was not present in the identical control reactionmixture. Fractions isolated from SEC-HPLC chromatography were analysedby SDS PAGE and the mono poly(ethylene glycol) conjugate was observed.

EXAMPLE 6 Polymer Conjugation to Asparaginase

A 100 μl sample of a 5 mg/ml solution of asparaginase at pH 6.5 (Sigma)was diluted with 900 μl of 20 mM phosphate buffer (pH 6 and alsocontaining 0.15 M NaCl and 5 mM EDTA). DL-dithioreitol (DTT, 15.4 mg)was then added and the resulting solution allowed to stand at roomtemperature. After allowing to stand for 2 h the solution was purifiedon a PD-10 column (Sephadex®G-25M, Pharmacia Biotech) that had beenequilibrated with 20 mM phosphate buffer (pH 6 and also containing 0.15M NaCl and 5 mM EDTA). The column was eluted with 1 ml portions of freshbuffer. Two fractions containing reduced protein were identified usingUV spectroscopy at 280 nm. These fractions were concentrated usingcentrifugal filter devices (MWCO 3,000; Microcon) to a volume ofapproximately 270 μl and then diluted to 1 ml with fresh pH 6 phosphatebuffer (additionally containing 0.23 mM hydroquinone). Two 5 μl sampleswere removed for later analysis by SDS PAGE. Separately, a 50 mg/mlsolution of the conjugating reagent solution ofα-methoxy-ω-4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzamide derivedfrom poly(ethylene glycol) (20,000 g/mol) was made up in the samephosphate buffer and placed in an ice-water bath along with the proteinsolution for 5 min. To the protein solution was then added 58 μl of theconjugation reagent solution and after several seconds of vortexing, thereaction solution was placed in a refrigerator (<5° C.). A controlreaction was also performed under the same conditions and scale butusing non-reduced asparaginase. Successful poly(ethylene glycol)conjugation to asparaginase was confirmed by SDS PAGE (4-12% Bis-TrisGel with colloidal blue staining) performed with samples taken from thereaction solution after 2 h. No conjugation band was observed by SDSPAGE for a sample taken from a control reaction where the asparaginasewas not allowed to react with DTT.

EXAMPLE 7 Polymer Conjugation to Interferon

A solution of 100 μl interferon α-2b (Shantha Biotechnics) (1 mg/ml)diluted with 150 μl of a buffer solution (sodium phosphate 0.02 M; NaCl0.15 M; EDTA 0.005 M; pH 6.0 in argon purged deionised water) waspartially reduced by the addition of selenocystamine (1 mM in argonpurged deionised water, 2 equivalents) and TCEP (1 mM in argon purgeddeionised water, 5 equivalents). The protein solution was cooled to 4°C. The conjugation reagentα-methoxy-ω-4-[2,2-bis[(p-tolylsulfonyl)methyl]acetyl]benzamide derivedfrom poly(ethylene glycol)(20,000 g/mol) was dissolved in the buffersolution (50 mg/ml), cooled to 4° C. and 16 μl added to the proteinsolution. The reaction mixture was vortexed and incubated at 4° C. for 2h. The non-ionic components of the reaction mixture were removed bycation exchange chromatography (Hitrap SP FF 1 ml column, Amershambiosciences) using two buffers: (A) 25 mM Na Acetate pH 4.0 and (B) 25mM Na Acetate+0.7 M NaCl pH 4.0. The column was washed (1 ml/min) withbuffer A (5.0 ml), then buffer B (5.0 ml) and then equilibrated with 10ml of buffer A (10 ml). The conjugate was loaded on the column (200 μl)followed by buffer A (500 μl) and a loading fraction (0.5 ml) wascollected. The column was washed with 5 ml of buffer A and fractionswere collected. The sample was eluted from the column with buffer B (10ml) and fractions were collected. Spectroscopy (UV, 280 nm) was used todetermine the fractions that contained protein and these fractions werethen purified into separate fractions by size exclusion chromatography(100 μl injections, superose 12-24 ml column, Amersham biosciences;eluent: 20 mM Na Phosphate, 0.15 M NaCl pH 7.0; isocratic elution for100 min at a flow of 0.25 ml/min; UV detection, 210 nm). Thesepurification conditions provided base line resolution for the separationof the poly(ethylene glycol) interferon conjugate from nativeinterferon. The conjugation reaction and purification of thepoly(ethylene glycol) conjugate were confirmed by SDS-PAGE; 12% Bis-Trisgel (SilverQuest Silver Staining; Colloidal Blue Staining and perchloricacid 0.1 M/BaCl₂ 5% and I₂ 0.1 M staining). No conjugation was observedwhen a solution of 40 μl of interferon (1 mg/ml) in 60 μl of sodiumphosphate 0.02 M, NaCl 0.15 M, EDTA 0.005 M, pH 6.0 in argon purgedwater was mixed with the conjugation reagentα-methoxy-ω-4-[2,2-bis[(p-tolylsulfonyl) methyl]acetyl]benzamide derivedfrom poly(ethylene glycol) (20,000 g/mol) was dissolved in the buffersolution (50 mg/ml), cooled to 4° C. and 16 μl added to the proteinsolution. This reaction mixture was vortexed and incubated at 4° C. for2 h.

The concentration of each purified fraction of the poly(ethylene glycol)interferon conjugate was determined by enzyme immuno assay. The sameassay was utilised to determine the concentration of the nativeinterferon that was conjugated and an international standard NIBSC (UK)sample of interferon. The reproducible and accurate standard curveobtained for all forms of the aforementioned interferon is shown inFIG. 1. A549 (human lung fibroblast) cells were plated at 15,000cells/well in 96-well flat-bottom tissue culture plates. Interferon fromthe NIBSC (UK), the native interferon that was conjugated and thepoly(ethylene glycol) interferon conjugate were then separately added tothe cells in triplicate and the plate incubated at 37° C. (5% CO₂) for24 h. On the next day, a working solution of EMCV (encephalomyocarditisvirus) was prepared in DMEM/2% FCS from stock virus stored at −80° C.The media containing the interferon or the poly(ethylene glycol)interferon conjugate was removed and replaced with media containingEMCV. The tissue culture plate was then incubated for 1 h at 37° C.after which the virus was removed. One hundred microliters of DMEM/10%FCS media was added and the plate incubated for 16-24 h at 27° C. From16 h onwards, the plate was read regularly to ascertain when cell deathwas starting. The media was then aspirated and the cells washed with 100μl of phosphate buffer solution. This was followed by the addition of 50μl of methyl violet solution [i.e., methyl violet (4% formaldehyde,0.05% methyl violet 2B (Sigma-Aldrich)] for 30 min at room temperature.The plate was then washed with 100 μl of water and dried in air.Spectrophotometric absorbance was determined at 570 nm using a platereader. FIG. 2 confirms that the interferon used for the conjugation hadequivalent activity of the international standard NIBSC (UK) interferon.FIG. 3 confirms that native interferon used for conjugation maintainedequivalent biological activity after being subjected to all the chemicaland purification processes except for exposure with the poly(ethyleneglycol) conjugation reagent. FIG. 4 confirms that the native interferonused for conjugation and the purified poly(ethylene glycol) interferonconjugate maintained equivalent biological activity.

Induction of 2′5′-oligoadenylate synthetase (2′5′-OAS) and proteinkinase R (PRKR) mRNA by interferon-α 2b was determined. The purifiedpoly (ethylene glycol) interferon conjugate and a sample of the nativeinterferon-α 2b were evaluated. Two million MOLT 4 cells/well wereincubated in 24 well tissue culture plates with 5000 pg of eachinterferon sample as measured using an enzyme immunoassay for 24 h at37°. The total RNA was extracted (RNA II Isolation kit, Macheray-Nagel)and 200 ng subjected to reverse transcription in a final volume of 20 μL(Sigma, AMV reverse transcription kit). Samples were diluted 1 in 4 inwater and 2 μL of each sample amplified in a 20 μL real-time PCRquantitation mix (Sigma SybrGreen ReadyMix). The primers used were:—

2′5′-OAS forward (SEQ ID NO: 1) GGC TAT AAA CCT AAC CCC CAA ATC2′5′-OAS reverse (SEQ ID NO: 2) AGC TTC CCA AGC TTC TTC TTA CAAPRKR forward  (SEQ ID NO: 3)  ACT CTT TAG TGA CCA GCA CAC TCGPRKR reverse  (SEQ ID NO: 4)  TTT AAA ATC CAT GCC AAA CCT CTT

For 2′,5′-OAS amplification, enzyme activation at 94° C. for 5 min wasfollowed by 48 cycles of denaturation at 94° C. for 5 sec, annealing at60° C. for 2 sec and extension at 72° C. for 8 sec. For PRKRamplification, samples were activated at 94° C. for 5 min and underwent48 cycles of denaturation at 94° C. for 5 sec, annealing at 59° C. for 2sec and extension at 72° C. for 8 sec. A melting curve analysis of theproducts was performed at the end of the amplifications. Quantitation ofinduced mRNA levels was done relative to the control untreated cellsusing the following formula:—Relative increase=2^(−(Ct sample−Ct control))where Ct is the threshold cross-over value.

FIG. 5, in which FIG. 5(a) shows the real-time quantitative RT-PCR(two-step) analysis of interferon-inducible 2′5′-OligoadenylateSynthetase (2′5′-OAS)gene, and FIG. 5(b) shows the real-timequantitative RT-PCR (two-step) analysis of interferon-inducible ProteinKinase R (PRKR) gene. The results confirm that the poly(ethylene glycol)interferon-α2b conjugate stimulated 2′5′-oligoadenylate synthetase(2′5′-OAS and protein kinase R (PRKR) mRNA synthesis to levels that weresimilar to native unpegylated interferon-α 2b.

The invention claimed is:
 1. A process which comprises reacting acompound of the general formula

in which one of X and X′ represents a polymer, and the other representsa hydrogen atom; Q independently represents a linking group; W′represents an electron-withdrawing group, or, if X′ represents apolymer, X-Q-W′ together represents an electron withdrawing group; Arepresents a C₁₋₅ alkylene or alkenylene chain; B represents a bond or aC₁₋₄ alkylene or alkenylene chain; and each L independently represents aleaving group; with compounds of the general formulae Z¹Nu and Z²Nu insuccessive steps, wherein Z¹ are Z² are separate proteins, and each Nuindependently represents an amine group, or with a compound of theformula Z(Nu)₂ in which Z represents a single protein, and each Nuindependently represents an amine group.
 2. A process as claimed inclaim 1, in which each leaving group L is independently —SR, —SO₂R,—OSO₂R, —N⁺R3, —N⁺HR2, —N⁺H₂R, halogen, or —OØ, in which R represents analkyl or aryl group and Ø represents a substituted aryl group containingat least one electron withdrawing substituent.
 3. A process as claimedin claim 1, in which the polymer is a polyvinylpyrrolidone, apolyacrylate, a polyoxazoline, a polyvinylalcohol, a polyacrylamide orpolymethacrylamide, a HPMA copolymer, a polyester, polyacetal,poly(ortho ester), polycarbonate, poly(iminocarbonate), polyamide, acopolymer of divinylether-maleic anhydride or styrene-maleic anhydride,a polysaccharide, or polyglutamic acid.
 4. A process as claimed in claim1, in which the polymer is a polyethylene glycol.
 5. A process asclaimed in claim 1, in which each linking group Q independentlyrepresents a direct bond, an alkylene group, or anoptionally-substituted aryl or heteroaryl group, any of which may beterminated or interrupted by one or more oxygen atoms, sulphur atoms,—NR groups in which R represents an alkyl or aryl group, keto groups,—O—CO— groups and/or —CO—O— groups.
 6. A process as claimed in claim 1,in which W′ represents a keto group, an ester group —O—CO— or a sulphonegroup —SO₂—.
 7. A process as claimed in claim 1, in which the compoundof formula II is reacted with a compound of the formula Z(Nu)₂ in whichZ represents a single protein, and each Nu independently represents anamine group.
 8. A process as claimed in claim 1, in which the compoundof formula (II) is reacted with compounds of the general formula Z¹Nuand Z²Nu in successive steps, wherein Z¹ are Z²are separate proteins,and each Nu independently represents an amine group.
 9. A process asclaimed in claim 2, in which the polymer is a polyvinylpyrrolidone, apolyacrylate, a polyoxazoline, a polyvinylalcohol, a polyacrylamide orpolymethacrylamide, a HPMA copolymer, a polyester, polyacetal,poly(ortho ester), polycarbonate, poly(iminocarbonate), polyamide, acopolymer of divinylether-maleic anhydride or styrene-maleic anhydride,a polysaccharide, or polyglutamic acid.
 10. A process as claimed inclaim 2, in which the polymer is a polyethylene glycol.
 11. A process asclaimed in claim 2, in which each linking group Q independentlyrepresents a direct bond, an alkylene group, or anoptionally-substituted aryl or heteroaryl group, any of which may beterminated or interrupted by one or more oxygen atoms, sulphur atoms,—NR groups in which R represents an alkyl or aryl group, keto groups,—O—CO— groups and/or —CO—O— groups.
 12. A process as claimed in claim 2,in which W′ represents a keto group, an ester group —O—CO— or a sulphonegroup —SO₂—.
 13. A process as claimed in claim 2, in which the compoundof formula II is reacted with a compound of the formula Z(Nu)₂ in whichZ represents a single protein, and each Nu independently represents anamine group.
 14. A process as claimed in claim 2, in which the compoundof formula (II) is reacted with compounds of the general formula Z¹Nuand Z²Nu in successive steps, wherein Z¹ are Z² are separate proteins,and each Nu independently represents an amine group.